Role of Endothelin Receptor B signaling in the genesis of aganglionic megacolon

描述（由申请人提供）：肠神经系统（ENS）主要由一组神经嵴衍生细胞（ENCC）发育而来，这些细胞在通过发育中的肠道向尾部前进之前进入咽部。 ENCC 在发育中肠道的进展需要它们的增殖、分化和迁移的协调。这些细胞过程中的任何一个单独或组合的缺陷都足以阻止 ENCC 的进展。当 ENCC 没有完成通过结肠的前进时，肠道的一个区域就会失去内在的神经支配，这就是先天性巨结肠症 (HD) 的代表。一组通常与 HD 相关的突变存在于编码配体 (EDN3) 或其受体内皮素-B (EDNRB) 的基因中。该受体在 ENCC 和肠道间质中表达。迄今为止，EDNRB 介导的 HD 的精确细胞缺陷和分子机制仍不清楚。为了解决这些问题，我们利用了一种新的条件 floxed 等位基因，它允许我们仅敲除神经嵴细胞中的 ednrb 表达。通过将我们的 floxed ednrb 小鼠与 Wnt1-Cre 转基因小鼠交配，我们特别删除了神经嵴衍生的 ENCC 中的 ednrb。此外，ENCC 还表达黄色荧光蛋白，从而可以记录它们的运动。我们的研究揭示了两种以前未被识别的与 EDNRB 信号传导缺陷相关的细胞缺陷。首先，在神经嵴细胞进入肠道后不久，空 ednrb 胚胎的 ENCC 进展就会延迟。其次，当无效的 ednrb ENCC 到达结肠时，它们表现出异常的轨迹和有缺陷的迁移。我们的数据表明这两个缺陷是相关的；最初的延迟导致 ENCC 比正常晚一天进入结肠。我们假设最初的延迟是由肠前神经嵴缺陷引起的，随后在后肠中无法前进是由于后肠环境的变化造成的。第一个具体目标是确定导致无效突变体中 ENCC 进展初始延迟的细胞缺陷。第二个目标是确定 ENCC 在结肠中迁移缺陷的机制。第三个具体目标是确定移植到无神经节的出生后后肠中的 ENCC 是否可以恢复后肠的协调运动功能。总之，这些研究将确定因缺乏 EDNRB 信号传导而导致 HD 的细胞缺陷，并确定利用移植的无效 ednrb ENCC 建立协调运动功能的可行性。公众健康相关性：我们的目标是阐明先天性巨结肠的机制，并设计一种为无神经节结肠区域提供功能性神经支配的方法。这些研究将使患有先天性巨结肠症和其他胃肠道运动障碍的患者受益，因为提供功能性神经支配是比手术切除更好的替代方案，手术切除会给患者带来终生的胃肠道问题。这里获得的信息也将与包括神经元在内的其他组织的移植相关。

DESCRIPTION (provided by applicant): The enteric nervous system (ENS) develops primarily from a cohort of neural crest- derived cells (ENCCs) that enter at the pharynx before advancing caudally through the developing gut. The advance of ENCCs through the developing gut requires their coordination of proliferation, differentiation, and migration. Defects in any of these cellular processes alone or combined are sufficient to stop ENCC advance. When ENCCs do not complete their advance through the colon, a region of the gut is left without intrinsic innervation and is representative of Hirschprung's disease (HD). One set of mutations commonly associated with HD is found in genes encoding the ligand (EDN3), or its receptor endothelin-B (EDNRB). The receptor is expressed in both ENCCs and gut mesenchyme. To date, the precise cellular defect and molecular mechanism responsible for EDNRB-mediated HD remain unknown. To address these issues we have utilized a novel conditional floxed allele that allows us to knockout ednrb expression in neural crest cells only. By mating our floxed ednrb mice with Wnt1-Cre transgenics we delete ednrb specifically in neural crest-derived ENCC. In addition, the ENCCs also express the yellow fluorescent protein, allowing their movement to be recorded. Our studies have revealed two previously unrecognized cellular defects related to defective EDNRB signaling. First, the ENCC advance in null ednrb embryos is delayed shortly after neural crest cells enter the gut. Second, as null ednrb ENCCs reach the colon, they display aberrant trajectories and defective migration. Our data indicate that these two defects are related; the initial delay results in ENCCs entering the colon a day later than normal. We hypothesize that the initial delay is caused by pre- enteric neural crest defect, and the subsequent failure to advance in the hindgut results from changes in the hindgut environment. The first specific aim is to determine the cellular defect responsible for the initial delay of ENCC advance in the null mutant. The second aim is to determine the mechanism for defective ENCC migration in the colon. The third specific aim is to determine whether ENCCs transplanted into the aganglionic postnatal hindgut can restore coordinated motor function to the hindgut. Together, these studies will identify cellular defects responsible for HD resulting from the absence of EDNRB signaling and determine the feasibility of establishin coordinated motor function with transplanted null ednrb ENCCs. PUBLIC HEALTH RELEVANCE: Our goals are to elucidate the mechanisms underlying Hirschsprung's disease and to devise a method to provide functional innervation to the region of aganglionic colon. These studies will benefit patients with Hirchsprung's disease and other gastrointestinal motility disorders because providing functional innervation is a superior alternative to surgical excision, which leaves patient with lifelonggastrointestinal problems. The information gained here will also be relevant to transplantation of other tissues including neurons.